Lipotropes (methionine, choline, folate, and vitamin B12) are dietary methyl donors and cofactors that are involved in one-carbon (methyl) metabolism (i.e., DNA methylation and nucleic acid synthesis). Studies have suggested that maternal methyl diet may affect the growth and health of the offspring via epigenetic changes in DNA methylation and gene expression in the offspring. However, the influence of prenatal dietary exposure to methyl nutrients on susceptibility to mammary carcinogenesis of the offspring is largely unknown. We postulate that in utero exposure to a lipotrope-additive diet may reduce mammary carcinogenesis in female offspring via epigenetic changes of gene expression. Methyl supplementation during pregnancy may increase one-carbon metabolism. Increased methyl metabolism may affect the expression of tumor-related genes and genes which are involved in DNA methylation pathways. This metabolic shift and altered gene signals consequently elicit a stable epigenetic imprint (memory) of gene expression, which may be passed on from dams to the developing fetus. Thus, prenatally imprinted gene signals could exert a protective effect against breast cancer development in the offspring. The overall goal of the proposed study is to establish a possible link between methyl diet-mediated and pregnancy-related epigenetic alteration of gene expression and reduction of mammary carcinogenesis in female offspring. The specific aim is to determine the extent to which prenatal exposure to methyl-supplemented diet decreases mammary carcinogenesis in offspring: it entails examining relationships between DNA methylation status and gene expression and susceptibility of the offspring to mammary carcinogenesis. Pregnant rats will be assigned to either the control or lipotrope-supplemented diet. At weaning, mammary tissues from dams will be collected to examine DNA methylation and gene expression. Half of the offspring will be used to collect mammary tissues for the same purposes as their dams (DNA methylation and gene expression). The remaining offspring will be utilized for the nitrosomethylurea-induced mammary carcinogenesis experiment, and tumor tissues will be collected. Mammary and tumor tissues will be analyzed for 1) global DNA methylation content by HPLC, 2) DNA methylation patterns of selected genes by methylation-specific PCR, and 3) expression of genes by quantitative RT-PCR and western blotting. Selected genes will include 1) those involved in DNA methylation pathways; DNA methyltransferase1, and 2) tumor- related genes; breast cancer1, p53, B-cell lymphoma2, methyl CpG binding protein2, and histone deacetylase1. Data from the proposed study may have clinical implications for development of maternal dietary strategies that decrease and possibly prevent breast cancer risk in female offspring. PUBLIC HEALTH RELEVANCE: The proposed study attempts to establish a link between methyl diet-mediated and pregnancy-related epigenetic changes of gene expression and susceptibility of offspring to mammary carcinogenesis. This study may provide the first evidence for the effect of in utero exposure to methyl nutrients on the reduction of susceptibility to breas cancer in female offspring. Data from the study may have clinical impacts for developing prenatal dietary strategies for reduction of breast cancer risk in offspring.